Establishment and maintenance of polarity in epithelial cells requires the assembly in basolateral and apical plasma membrane domains of discrete subsets of proteins responsible for the specialized functions at the cell surfaces including directional ion currents across the epithelium, directional transport of nutrients, and receptor-mediated signal transduction. The dynamics of plasma membrane composition require, not only directional trafficking of newly synthesized proteins to their correct surface, but also coordination of protein internalization, recycling and transcytosis between membrane surfaces. Recent evidence suggests that Rab11 small GTPase family members (Rab11a and Rab25) associated with the recycling system regulate the establishment and maintenance of polarity. In addition, members of a family of Rab11 interacting proteins (Rab11-FIPs) modulate discrete aspects along the recycling system pathway. In particular, expression of mutants of Rab11-FIP2, which coordinates a complex with both Rab11a and myosin Vb, can inhibit transcytosis in polarized epithelial cells. Furthermore, expression of a point mutant of FIP2 that abolishes phosphorylation of FIP2 by MARK2/Par1b causes a delay in the establishment of polarity by polarized MDCK cells. All of these studies have pointed to the importance of Rab11-FIP proteins in the interaction of recycling system elements with the microtubule cytoskeleton. Alterations in these trafficking pathways and cell polarity are also intimately connected to changes that lead to transformation and enhanced migratory phenotypes. Thus, in preliminary findings, we have demonstrated that genetic deficiency in the epithelial specific small GTPase Rab25 leads to enhanced intestinal and colonic adenoma formation in ApcMin mice. These findings support the concept that specific trafficking pathways are critical to the proper maintenance of normal polarized phenotypes and confer a tumor suppressor function in gastrointestinal epithelial cells. We have hypothesized that the Rab11 family members, in concert with their associating interacting proteins, Rab11-FIPs, perform critical roles in maintaining the polarized cell phenotype. As a corollary to this hypothesis, we propose that perturbation of specific aspects in these vesicle trafficking pathways can regulate both plasma membrane composition and differentiated phenotype, in some cases leading to transformation. To examine aspects of this hypothesis, we will first determine the regulation of Rab11-FIP protein structures and the structural requirements for assembly of Rab11-FIP protein coordinated complexes. Second, we will examine the roles of the microtubule cytoskeleton and the association of Rab11-FIP proteins with microtubules in regulating the plasma membrane recycling system and polarized trafficking. Third, we will determine the role of Rab11 family coordinated complexes in the regulating epithelial polarity and transformation. All of these studies will allow us to continue the examination of the role of recycling system components in the dynamic regulation of the mechanisms required for the establishment and maintenance of the polarized cell phenotype. PUBLIC HEALTH RELEVANCE: This proposal focuses on the mechanisms regulating the ability of epithelial cell to maintain distinct apical and basal domains. Proteins are regulated at these surfaces by correct trafficking and recycling of internalized proteins back to correct membrane surfaces. Loss of proper trafficking can lead to the inadequate barrier function, inappropriate secretion or even to transformation and the development of cancer.